The present invention is directed to compositions involved in cell cycle regulation and methods of use. More particularly, the present invention is directed to genes encoding proteins and proteins involved in cell cycle regulation, particularly those having homology to tankyrase. Methods of use include use in assays screening for modulators of the cell cycle and use as therapeutics.
Cells cycle through various stages of growth, starting with the M phase, where mitosis and cytoplasmic division (cytokinesis) occurs. The M phase is followed by the G1 phase, in which the cells resume a high rate of biosynthesis and growth. The S phase begins with DNA synthesis, and ends when the DNA content of the nucleus has doubled. The cell then enters G2 phase, which ends when mitosis starts, signaled by the appearance of condensed chromosomes. Terminally differentiated cells are arrested in the G1 phase, and no longer undergo cell division.
The hallmark of a malignant cell is uncontrolled proliferation. This phenotype is acquired through the accumulation of gene mutations, the majority of which promote passage through the cell cycle. Cancer cells ignore growth regulatory signals and remain committed to cell division. Classic oncogenes, such as ras, lead to inappropriate transition from G1 to S phase of the cell cycle, mimicking proliferative extracellular signals. Cell cycle checkpoint controls ensure faithful replication and segregation of the genome. The loss of cell cycle checkpoint control results in genomic instability, greatly accelerating the accumulation of mutations which drive malignant transformation. Thus, modulating cell cycle checkpoint pathways and other such pathways with therapeutic agents could exploit the differences between normal and tumor cells, both improving the selectivity of radio- and chemotherapy, and leading to novel cancer treatments. As another example, it would be useful to control entry into apoptosis.
It is also sometimes desirable to enhance proliferation of cells in a controlled manner. For example, proliferation of cells is useful in wound healing and where growth of tissue is desirable. Thus, identifying modulators which promote, enhance or deter the inhibition of proliferation is desirable.
Continuous cell proliferation, as in cancer, requires the replication of DNA including chromosome ends known as telomeres. Without being bound by theory, it appears that telomeres decrease in size with successive cell divisions, that the number of divisions a cell is capable of negatively correlates with telomere length, and that a cell cannot divide further once a critical telomere length has been reached. Further, the normal process of telomere shortening with successive cell divisions appears to be circumvented in cancer, suggesting the maintenance of telomore length may be critical to normal and oncogenic growth.
Without being bound by theory, it appears that the synthesis of telomeres involves unique DNA replication mechanisms. These mechanisms act to extend telomeres prior to cell division, and are critical to the determination of telomere length in daughter cells. Several molecules involved in telomere synthesis have been identified, including the proteins telomerase, TRF-1 and tankyrase. These and other molecules involved in telomere synthesis provide unique targets for intervention strategies designed to modulate cell proliferation.
Recognized in this application is the hypothesis that these two aspects of cell proliferation control, namely check point modulation and telomere maintenance, intersect in some aspect. The present application sets forth tankyrase h nucleic acids and proteins which, without being bound by theory, appear to bridge the gap that currently exists between these two points of control.
Despite the desirability of identifying cell cycle components and modulators, there is a deficit in the field of such compounds. Accordingly, it would be advantageous to provide compositions and methods useful in screening for modulators of the cell cycle. It would also be advantageous to provide novel compositions which are involved in the cell cycle.
The present invention provides cell cycle proteins and nucleic acids which encode such proteins. Also provided are methods for screening for a bioactive agent capable of modulating the cell cycle. The method comprises combining a cell cycle protein and a candidate bioactive agent and a cell or a population of cells, and determining the effect on the cell in the presence and absence of the candidate agent. Therapeutics for regulating or modulating the cell cycle are also provided.
In one aspect, a recombinant nucleic acid encoding a cell cycle protein comprising a nucleic acid that hybridizes under high stringency conditions to a sequence as set forth in FIG. 1 or FIG. 2 (SEQ ID NOS:1-2), or their complements, is provided. In a preferred embodiment, the recombinant nucleic acid encodes a protein which binds to p21.
In another aspect, a recombinant nucleic acid encoding a cell cycle protein comprising a nucleic acid having at least 85% sequence identity to a sequence as set forth in FIG. 1 or FIG. 2 (SEQ ID NOS:1-2) is provided. In one embodiment, a recombinant nucleic acid is provided which encodes a polypeptide having the sequence as shown in FIG. 3 or FIG. 4 (SEQ ID NOS:3-4). In yet another aspect, a recombinant nucleic acid is provided herein which has a nucleic acid sequence as set forth in FIG. 1 or FIG. 2.
In a further aspect, expression vectors are provided herein. In one embodiment, the vector comprises any one of the recombinant nucleic acids described herein, operably linked to regulatory sequences recognized by a host cell transformed with the nucleic acid. Moreover, host cells comprising any one of the nucleic acids or vectors described herein are provided.
Also provided herein is a process for producing a cell cycle protein comprising culturing any one of the host cells described herein under conditions suitable for expression of a cell cycle protein. In one embodiment, the cell cycle protein is recovered.
In a further aspect herein, a recombinant cell cycle protein encoded by any one of the nucleic acids described herein is provided. In yet another aspect, a recombinant polypeptide comprising an amino acid sequence having at least 85% sequence identity with the sequence set forth in FIG. 3 or FIG. 4 (SEQ ID NOS:3-4) is provided herein. In one embodiment, the recombinant polypeptide provided herein bind to p21. In another embodiment, the recombinant polypeptide provided herein has a sequence as set forth in FIG. 3 or FIG. 4.
Also provided herein is an isolated polypeptide which specifically binds to a cell cycle protein. In one aspect, the polypeptide is an antibody. In a preferred embodiment, the antibody is a monoclonal antibody. In another preferred embodiment, the antibody reduces or eliminates the biological function of said cell cycle protein.
In a further aspect, the present invention provides a method for screening for a bioactive agent capable of binding to a cell cycle protein tankyrase H. In a preferred embodiment, said method comprises combining a cell cycle protein tankyrase H and a candidate bioactive agent, and determining the binding of said candidate agent to said cell cycle protein tankyrase H.
In one embodiment, a method for screening for agents capable of interfering with the binding of a cell cycle protein tankyrase H and p21 is provided. In a preferred embodiment, a screening method as described herein comprises combining a cell cycle protein tankyrase H, a candidate bioactive agent and a p21 protein, and determining the binding of said cell cycle protein and said p21 protein. In one case, said cell cycle protein and said p21 protein are combined first.
In yet another embodiment a method for screening for a bioactive agent capable of modulating the activity of a cell cycle protein tankyrase H is provided. In one aspect, the method comprises the steps of adding a candidate bioactive agent to a cell comprising a recombinant nucleic acid encoding a cell cycle protein tankyrase H and determining the effect of the candidate bioactive agent on said cell. In another embodiment, a library of candidate bioactive agents are added to a plurality of cells comprising a recombinant nucleic acid encoding a cell cycle protein.
Also provided herein is a method of diagnosing cancer. In one embodiment, the method comprises determining the level of expression of tankyrase H from a test sample of an individual and comparing the level with a control which indicates there is no cancer, wherein an increase in the level of expression of tankyrase H in the test sample over the control sample indicates that the individual has cancer. In another embodiment, the method comprises determining the activity of tankyrase H, preferably poly ADP-ribose polymerase (PARP) activity, in a test sample from an individual and comparing the level with a control. An alteration (eg. an increase) in the activity of tankyrase H in the test sample over the control sample indicates that the individual has cancer.
Further provided herein are methods for the treatment of individuals affected by dysfunction of tankyrase activity, tankyrase H activity, telomerase activity, or cancer, using a pharmaceutical composition comprising modulators of tankyrase H activity, including antisense oligonucleotides and bioactive agents capable of binding to and/or modulating the activity of tankyrase H.